Cigarette smoking is a major independent risk factor for cardiovascular (CV) disease. One early event that contributes to this risk is endothelial dysfunction, resulting from the loss of endothelial-derived vasodilators. Endothelial dysfunction precedes many vascular diseases and occurs prior to symptoms of overt clinical disease in cigarette smokers. It is notable that cigarette smoking is the single most important factor that reduces omega- 3 polyunsaturated fatty acids (n-3 PUFA), including eicosapentaenoic and docosahexaenoic acids (EPA & DHA), and that n-3 PUFA supplementation can normalize endothelial dysfunction in cigarette smokers. However, the mechanism by which n-3 PUFAs reverse endothelial dysfunction is not known. Our preliminary data show that the EPA and DHA epoxide metabolites are potent vasodilators via nitric oxide (NO) and potassium (K+) channels. Our preliminary data also show that a DHA epoxide metabolite activates antioxidant defense mechanisms regulated by Nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Finally, our recent data show that n-3 PUFAs are highly correlated with endothelial dysfunction in healthy young cigarette smokers. Thus, we hypothesize that n-3 PUFAs improve cigarette smoke (CS)-induced endothelial dysfunction by preserving vasodilation and enhancing antioxidant responses via their epoxide metabolites. To test this, in Aim 1 we will determine the mechanism by which n-3 PUFA epoxides prevent CS-induced endothelial dysfunction. We will increase n-3 PUFA epoxides by increasing endogenous production of n-3 PUFAs using a transgenic mouse that expresses invertebrate n-3 fatty acid desaturase-1 (fat-1), and by direct administration via osmotic pump. Mice will then be exposed to filtered air or mainstream CS, and we will elucidate the mechanisms by which epoxides prevent endothelial dysfunction in aorta and mesenteric arterioles. In Aim 2, we will determine the mechanism by which n-3 PUFA epoxides reduce CS-induced vascular oxidative stress. Aortic vessels will be incubated ex vivo with DHA epoxides and an oxidative stress/antioxidant defense pathway PCR array used as an unbiased screen for assessing vascular antioxidant responses. We also will use the mouse models in aim 1 exposed CS, and assess systemic antioxidant capacity, oxidative stress indices, and RNA and protein expression of genes identified from the PCR array, including Nrf2-regulated genes. In Aim 3, we will translate our basic science studies into the clinic to elucidate the ability of n-3 PUFA epoxides to serve as biomarkers of oxidative stress and endothelial dysfunction in never and current cigarette smokers. We will assess n-3 PUFAs and their epoxide metabolites, systemic antioxidant capacity and oxidative stress indices, RNA expression of Nrf2-regulated antioxidant genes and antioxidant polymorphisms. We will measure brachial artery flow-mediated dilation (FMD) and classic CV disease risk factors. n-3 PUFAs, their epoxides, and CV risk factors will be correlated individually with oxidative stress and FMD, and significant variables used in a multivariate regression model to predict oxidative stress and FMD.